Expression-enhanced polypeptides

ABSTRACT

A composite polypeptide, said composite polypeptide comprising a desired polypeptide and an expression enhancing domain (“EED”), said EED comprising first and second cysteine amino acid residues Cys1 and Cys2, respectively, Cys1 being located closer to the N-terminus of the composite polypeptide molecule than Cys2, wherein Cys1 and Cys2 are separated by a polypeptide linker, said linker—being free of cysteine and proline;—defining a length sufficient to allow Cys1 and Cys2 to engage in an intramolecular disulfide bond with one another; and—having a flexible polypeptide conformation essentially free of secondary polypeptide structure in aqueous solution, wherein at least one of Cys1 and Cys2 is derivatized with a derivatization moiety.

This application is a national phase application under 35 U.S.C. §371 ofInternational Application No. PCT/EP 2005/007748 filed 15 Jul. 2005,which claims priority to European Patent Application No.: EP 04 016890.8 filed 16 Jul. 2004. The entire text of each of theabove-referenced disclosures is specifically incorporated herein byreference without disclaimer.

The invention relates to polypeptide molecules which have been modifiedto improve their expression characteristics. The modified polypeptidemolecules are expressed in better/higher yields than their correspondingpartners, i.e., polypeptide molecules that have not been modified (onthe nucleic acid level). The invention further relates to compositionscomprising such polypeptides. Finally, the invention provides a methodto prepare the modified polypeptide molecules mentioned previously.Throughout the following description, mention of a (composite)polypeptide is to be understood as implying both the polypeptide per seand, where appropriate, the corresponding nucleic acid sequence, as willbe appreciated by the skilled reader. The same applies for the (desired)polypeptide and the expression-enhancing domain (EED).

Expression of recombinant polypeptides in microbial host systems is anefficient way of producing large amounts of a desired polypeptide. Whenthe polypeptide produced is intended for use as a diagnostic and/ortherapeutic agent, further modification of the polypeptide as expressedis often necessary. For example, a polypeptide intended for use as adiagnostic agent might need to be modified such that it can bind to asolid support. Alternatively, the polypeptide may need to be coupled toan agent allowing it to be visualized by a certain imaging method. Apolypeptide intended for administration to a patient as part of a courseof therapy may need to be modified in order to modulate its in vivoproperties, for example its pharmacokinetic properties.

Derivatization of a recombinantly produced polypeptide is most oftenaccomplished by chemical reaction between a chemical substance(“derivatization moiety”) and a reactive side group of one or more ofthe amino acids comprised within the polypeptide. The result is acovalent coupling of the derivatization moiety to the polypeptide,wherein the location and valency of such coupling(s) is dictated by therespective location and number of reactive amino acids within thepolypeptide. This means that in a polypeptide with multiple reactiveamino acids, chemical coupling of a derivatization moiety with thepolypeptide will occur multiple times at locations throughout thepolypeptide corresponding to the locations of the reactive amino acids.

For some purposes, such a multivalent, site-nonspecific coupling ofderivatization moieties to a polypeptide may be desirable, but moreoften it is not. For example, in a diagnostic procedure, it may beimportant for accurate quantification of measurements to limit thenumber of derivatizations per polypeptide molecule to one. Similarly,successful therapeutic use of a polypeptide in vivo often hinges on theability of the medical practitioner to precisely predict and control thebiological activity of this polypeptide. In such a situation, variationsresulting from uncontrolled and site-nonspecific derivatization of thepolypeptide used may understandably be inconsistent with the intendedcourse of therapy. In addition, a site-nonspecific coupling of atherapeutic or diagnostic polypeptide with a derivatization moiety maylead to an impairment of the polypeptide's desired activity. This mightfor example be the case when a single chain antibody polypeptide isderivatized in a site-nonspecific fashion such that the antigen bindingsite is sterically and/or electrostatically prevented from binding toantigen or reduced in its binding activity. In such a case, the desiredtherapeutic or diagnostic effect of the single chain antibody may beabolished or at least attenuated.

It is often desirable, then, to engineer recombinant polypeptides suchthat derivatization is possible only at predefined locations, or only atone predefined location in the polypeptide molecule. The valency ofcoupling can be tuned by controlling the number of reactive amino acidsin the polypeptide, and the desired polypeptide activity and/or chemicalcharacteristics may be modulated by planning the location of suchcouplings so as not to physically impair the interaction of thepolypeptide with other molecules in the environment.

One amino acid which has proven useful in this regard is cysteine.Because of its importance in stabilizing protein structure via formationof disulfide bonds, cysteine normally occurs in polypeptides only atdefined locations. By incorporating a single cysteine into a “benign”region of the polypeptide not directly required for the desiredpolypeptide activity, one can take advantage of cysteine's reactivesulfhydryl side chain as a natural anchor point for a desiredderivatization without, or without significantly affecting the desiredpolypeptide activity (Volkel T., et al. (2004) Biochim Biophys Acta1663, 158-66).

However, incorporation of additional cysteine residues into polypeptidesfor purposes of derivatization entails certain disadvantages. Often, thedesired polypeptide will already have cysteine residues in its aminoacid sequence for the purposes of structural stabilization. Anadditional cysteine incorporated for the purpose of derivatizing thepolypeptide with a derivatization moiety may in this case enter intoundesirable disulfide linkages with such already present cysteines,severely perturbing the polypeptide structure necessary for a desiredactivity.

Even if the desired polypeptide does not itself contain any cysteineresidue in its amino acid sequence, the incorporation of a singlecysteine residue can still lead to problems. Following expression in ahost organism, polypeptides containing an engineered cysteine amino acidresidue can form polypeptide dimers with one another via intermoleculardisulfide bonds between the thiol (i.e. sulfhydryl) groups of the twocysteine residues in the respective polypeptides (Albrecht H., et al.(2004) Bioconjug Chem 15, 16-26; Olafsen T., et al. (2004) Protein EngDes Sel 17, 21-7). This danger is particularly large when usingprokaryotic expression systems to produce the polypeptide. This isbecause in such systems, proteins are gradually transported into theperiplasmatic space of the microbial host, where oxidative conditionsprevail While such oxidative conditions are essential for the formationof desirable, structure-stabilizing disulfide bonds in the nascentpolypeptide chain, they also promote the formation of undesirableintermolecular disulfide bonds between free cysteine residues intendedas later derivatization points in two respective polypeptides.

The above issues are not limited to expression in prokaryotes. In Luo etal. (1997) J Biochem 121, 831-4, experiments were described comparingthe amount of yeast-expressed monomeric and dimeric (i.e. linked via anintermolecular disulfide bond) scFv polypeptide depending on whetherthis scFv polypeptide comprised one or two C-terminal cysteine residues.It was found that scFv with a single C-terminal cysteine residue wasmore likely to exist in dimeric form, while scFv with two C-terminalcysteine residues was more likely to exist in monomeric form. It is alsoapparent from this publication that the total amount of expressedpolypeptide remains about the same, irrespective of the isoformdistribution. Additionally, it is revealed that the construct that hastwo cysteine residues (which exhibits a tendency to form anintramolecular disulfide bond) exhibits only poor binding activity.

Especially when expressing polypeptides intended for therapeutic use, itis often important for reasons of product homogeneity to produce themonomeric rather than the dimeric isoform. The prior art, embodied bythe above mentioned publication of Luo et al., then, provides theresearcher interested in expressing a monomeric polypeptidederivatizable at cysteine with certain tools to achieve this end.However, the prior art does not provide for a tool suitable to expressthe (monomeric) isoform in acceptable amounts and with acceptablebinding activity. Thus, it was an object of the present invention todevelop a DNA construct allowing high yield expression of thecorresponding polypeptide exhibiting an acceptable binding activity,wherein the polypeptide is predominantly obtained as a monomericpolypeptide.

The inventors have solved this object by providing a nucleic acidencoding a so-called composite polypeptide according to the presentinvention, as defined in the claims. The nucleic acid, if appropriatelyexpressed, provides for a composite polypeptide, said compositepolypeptide comprising a desired polypeptide and an expression enhancingdomain (“EED”), said EED comprising first and second cysteine amino acidresidues Cys1 and Cys2, respectively, Cys1 being located closer to theN-terminus of the recombinant polypeptide molecule than Cys2, whereinCys1 and Cys2 are separated by a polypeptide linker, said linker

-   -   being free of cysteine and proline;    -   defining a length sufficient to allow Cys1 and Cys2 to engage in        an intramolecular disulfide bond with one another; and    -   having a flexible polypeptide conformation essentially free of        secondary polypeptide structure in aqueous solution,        wherein at least one of Cys1 and Cys2 is derivatized with a        derivatization moiety.

By incorporating not one but two cysteine residues Cys1 and Cys2 intothe EED and by tuning the length and specifying the nature of thepolypeptide linker sequence disposed therebetween to promote formationof an intramolecular disulfide bond between Cys1 and Cys2, such adisulfide bond forms, rendering Cys1 and Cys2 unable to participate inunwanted inter—or intramolecular disulfide bridges as described above.In a sense, each of Cys1 and Cys2 become the respective other'sprotective group. When an intramolecular disulfide loop has been formedbetween Cys1 and Cys2, Cys2 may be seen as the derivatizing moiety ofCys1. Conversely, Cys1 may be seen as the derivatizing moiety of Cys2.

Composite polypeptides which have been engineered, at the nucleic acidlevel, to contain two cysteine residues as described above have theadvantage that they bear chemical anchor points for later (i.e.post-expression and -isolation) derivatizations. At the same time, thedanger of formation of unwanted intermolecular disulfide linkages isdrastically reduced, as such danger would in this case arise mainly fromother cysteine residues present in the desired polypeptide for the(desirable) disulfide stabilization of polypeptide structure. Suchdisulfide bonds normally form in an oxidative environment during and/orfollowing translation as the nascent polypeptide gradually grows. Assuch, any free sulfhydryl group of a cysteine needed for thestabilization of polypeptide structure normally finds its disulfidepartner relatively quickly and is thus blocked from further unwantedreactions. The incorporation of a linker optimized in length, chemicaland steric properties to allow Cys1 and Cys2 to form a mutual disulfidebond ensures that Cys1 and Cys2 will react only with one another and notwith a spatially distant cysteine residue in the polypeptide which isneeded for stabilization of polypeptide structure, but which has not yetreacted with its intended counterpart cysteine residue. In short, thelinker ensures that Cys1 and Cys2 will always be closer to one anotherthan either Cys1 or Cys2 is to any other cysteine residue in thepolypeptide.

Once expressed and isolated, such a composite polypeptide exhibiting adisulfide bond between Cys1 and Cys2 may be exposed to reducingconditions sufficient to open (only) the disulfide linkage between Cys1and Cys2. Following this, derivatization of Cys1 and/or Cys2 withderivatization moieties other than the respective other Cys1 or Cys2 maybe performed.

In addition to the advantage of obtaining a derivatized polypeptide(which can be rederivatized following isolation) without thedisadvantages described above, it has also surprisingly been found thatpolypeptides which have been engineered to comprise an EED (i.e.,composite polypeptides within the meaning of the invention) also exhibithigher levels of overall expression from their corresponding nucleicacids as compared to polypeptides which do not comprise an EED. Whilethe composite polypeptide according to the invention may be expected toresult in less unwanted dimer than a polypeptide without the EED, it isentirely unexpected based on the teaching in the prior art (e.g., thepublication of Luo et al., cited above) that the overall expression oftotal polypeptide with acceptable binding activity would be increased byincorporation of the EED according to the present invention. Thecomposite polypeptide of the invention, then, can be produced in overallhigher yield as compared to the desired polypeptide without EED, whereinthe ratio of monomeric composite polypeptide relative to dimericcomposite polypeptide is increased relative to the ratio seen inproduction of the desired polypeptide lacking an EED. In this way,monomeric polypeptide is not only favored over dimeric polypeptide, butthe overall higher amount of polypeptide results in much more (over5-fold more) monomeric polypeptide which can then be (re-)derivatized asneeded.

Without being bound by theory, the inventors believe that the surprisingincrease in total expressed polypeptide observed is due at least in partto the propensity of Cys1 and Cys2 within the EED to form a disulfidebond with one another. To explain why this is believed to be so, it ishelpful to consider what happens during the subsequent expression of twoidentical polypeptides which do not comprise an EED as defined above.For the purposes of the foregoing discussion, these identicalpolypeptides will be referred to as PP1 and PP2, and each comprises thesame desired polypeptide as well as a portion C-terminal thereto withonly one cysteine residue (i.e. neither PP1 nor PP2 comprises an EED asdefined above). The descriptors 1 and 2 denote, then, not differentpolypeptide identities, but rather the chronological order in whichidentical polypeptides are expressed.

Considering now that PP1 is expressed before PP2, it (PP1) willgradually be transported into an oxidative cellular environment in thedirection N-->C, meaning that the amino terminus—that of the desiredpolypeptide—will be the first end to emerge into said oxidativeenvironment. Emerging in this way, a cysteine residue within the desiredpolypeptide which will participate in a structure-stabilizing disulfidebond need only wait for its partner cysteine residue, the latter beinglocated more towards the C-terminal end of the desired polypeptide—toemerge into the oxidative environment for said disulfide bond to form.This process continues as the desired polypeptide continually emerges,until all disulfide bonds necessary for structural stabilization withinthe desired polypeptide have been formed. Once the desired polypeptidecomponent of PP1 has completely emerged (and properly folded), theC-terminal portion of PP1 with only one cysteine residue emerges.However here, there exists no partner cysteine with which this singlecysteine may react to form a disulfide bond, so this single cysteineresidue remains unpaired. Once the finished PP1 has been released, then,the desired polypeptide portion of PP1 is properly folded anddisulfide-stabilized, and the C-terminal portion of the molecule bearsone cysteine residue with a reactive sulfhydryl group.

Considering now that PP2 begins to be expressed into the sameenvironment in which the completed PP1 resides, the N-terminal end ofPP2 will first emerge. The first cysteine residue within the desiredpolypeptide portion of PP2 emerges but cannot yet form the intendedstructure-stabilizing disulfide bond since its cysteine reaction partnerwithin the desired polypeptide portion of PP2 has not yet emerged.However, the single, unpaired cysteine residue within the desiredpolypeptide portion of PP2 may react with the single, unpaired cysteineresidue in the C-terminal portion of the already completed PP1. In thisway, an unwanted disulfide bond is formed between the first cysteineresidue within the desired polypeptide portion of PP2 and the unpairedcysteine residue in the C-terminal portion of PP1. In such a scenario,the second cysteine residue in the desired polypeptide portion of PP2would react with the unpaired cysteine residue in the C-terminal portionof PP2, or another cysteine residue within the desired polypeptideportion of PP2. Either way, the array of resulting disulfide bonds willvery likely result in an improperly assembled polypeptide complexdevoid, or substantially devoid of the biological activity of thedesired polypeptide.

Such an improperly assembled polypeptide is likely to be recognized assuch and degraded by intracellular proteinases, thus reducing the amountof total polypeptide obtained. In the event that such an improperlyassembled polypeptide is not actively degraded in this manner, it islikely to exist as an insoluble aggregate with other malformedpolypeptides of this type, and would be removed from properly assembledpolypeptide in the course of standard polypeptide isolation procedures.In any event, polypeptide which is improperly assembled in this mannerwill tend to lower the amount of properly assembled polypeptide finallyobtained in standard polypeptide isolation procedures.

In contrast, a composite polypeptide according to the inventioncomprises not only two cysteine residues (Cys1 and Cys2) in an EED, butalso a linker disposed therebetween, said linker having been optimizedto promote disulfide bond formation between Cys1 and Cys2. In light ofthe above considerations regarding PP1 and PP2, and supposing now thatPP1 and PP2 are composite polypeptides according to the invention (i.e.they each comprise an EED), it is clear that neither Cys1 nor Cys2 ineach of the respective EEDs will remain unpaired, since Cys1 and Cys2within a respective EED will have formed a disulfide bond with oneanother. Improperly assembled polypeptides are avoided, having theeffect that the products are not degraded by intracellular proteinasesand/or do not form aggregates. As a result, the amount of compositepolypeptide expressed and isolated is greatly increased.

In summary, then, the expression of the composite polypeptide accordingto the invention results in an increase in the monomer:dimer ratio ofpolypeptide. At the same time, the levels of overall polypeptideobtained—regardless of the isoform of the polypeptide—are also increasedrelative to a polypeptide lacking the EED as defined above. The netresult is that a very small amount of dimeric and/or multimericpolypeptide and a vastly larger amount of monomeric polypeptide isobtained with a composite polypeptide comprising the EED as describedabove than is observed for comparative desired polypeptides in which anEED as defined above is lacking.

Within the meaning of the present invention, “N-terminus” and“C-terminus” are to be understood according to established convention inbiochemistry: The N-terminus of a polypeptide is the end of thepolypeptide chain ending in an amino group, while the C-terminus of apolypeptide is the end of the polypeptide chain ending in a carboxylgroup. The fact that Cys1 is located closer to the N-terminus than Cys2establishes the orientation of Cys1 and Cys2 relative to one anotherwithin the polypeptide chain. By this, the orientation of the EED inwhich Cys1 and Cys2 are comprised is also established.

Within the meaning of this embodiment of the invention, a polypeptidelinker with a “flexible polypeptide conformation” is a polypeptidelinker having at each covalent bond within the polypeptide chainsufficient degrees of rotational freedom to render the polypeptidelinker as a whole largely unrestricted, i.e. restricted only by itslength, in the conformations it may assume within three-dimensionalspace. As such, imagining a polypeptide linker anchored at one end at animaginary point in three-dimensional space and defining a sphere aroundthis point with a radius corresponding to the length of the fullyextended polypeptide linker, if the polypeptide linker has a “flexiblepolypeptide conformation”, the distal end of this polypeptide linker(i.e. the free, non-anchored end) must be able to touch any point inthree-dimensional space located on or within said sphere with equalease. This model gives rise to the corollary that such a polypeptidelinker with “flexible polypeptide conformation” must also be“essentially free of secondary polypeptide structure”, for example ofstretches of alpha-helix or beta-sheet. Any predisposition of thepolypeptide linker toward a motif of polypeptide secondary structurewill necessarily limit the degree of spatial freedom enjoyed by thelinker's free end, thereby constraining this end with regard to thepoints it is able to reach within the sphere defined above. Thisflexibility contributes to the ability of the linker to double back onitself, thereby allowing Cys1 and Cys2 to form an intramoleculardisulfide linkage.

Undesired secondary structure may be ordered (as in the case of thealpha-helix and beta sheet described above) or may be disordered, aswould be expected if, say, a proline residue were to exist in the linkersequence (the constrained ring in proline is known to cause kinks in thepolypeptide backbone). Without being bound by theory, the inventorsbelieve that this intrinsic flexibility of the linker between Cys1 andCys2 is a major determinant in ensuring the formation of a disulfidebond between these two cysteine residues, and that efficient disulfidebond formation is linked to the marked enhancement of overallpolypeptide expression observed (see reasons as set out above). For thisreason it is important to avoid including amino acids such as proline inthe sequence of the linker, since such incorporation restricts the freemovement of the linker necessary to allow Cys1 and Cys2 to migrate intoone another's vicinity such that the desired disulfide bond forms. Aminoacid residues that are allowed in the linker according to the presentinvention comprise, but are not limited to, Gly, Ala, Val, Leu, Ile,Ser, Thr, Met, Tyr, Asn, Gln.

Within the meaning of the present invention, the term “derivatized” isto be understood as describing a situation in which one or both of theamino acid residues Cys1 and Cys2 have entered into reaction with a“derivatization moiety”. A derivatization moiety may for example be acompound comprising a maleimide group, for example a PEG moleculecomprising a maleimide group. In this particular exemplary, non-limitingcase, the resulting derivatized Cys1 and/or Cys2 will have beenderivatized with a PEG molecule via a covalent S-C bond resulting fromnucleophilic attack by the sulfur atom in Cys1 and/or Cys2 with one ofthe unsaturated carbon atoms within the maleimide ring. As anotherexample, a “derivatization moiety” may also be a molecule which itselfcomprises a sulfhydryl group, so that the resulting derivatized Cys1and/or Cys2 will have been derivatized with this molecule via a covalentS-S, i.e. disulfide, bond. It is within the meaning of “derivatized”that Cys1 and/or Cys2 can react with another cysteine residue in thesame or another polypeptide chain. Specifically, the formation of thedesired intramolecular disulfide bridge between Cys1 and Cys2 asdescribed above is to be understood as falling within the meaning of“derivatized” in the present invention; in this case, Cys1 will havebeen derivatized by Cys2, and vice versa. Generally, then, from thestandpoint of Cys1, “derivatized” covers all scenarios in which Cys1 hasparticipated in a covalent chemical reaction with a species other thanitself (e.g. with Cys2). Likewise, “derivatized” also covers allscenarios in which Cys2 has participated in a covalent chemical reactionwith a species other than itself (e.g. with Cys1).

According to a preferred embodiment of the invention, at least 75% ofthe amino acid residues in the. linker are selected from Gly, Ala, Val,Leu, Ile, Ser, Thr, Met, Tyr, Asn, and Gln.

Most preferred are Gly, Ser, Ala and Thr. These amino acids are eitheruncharged, are well or reasonably well soluble in water, or are both.

According to a preferred embodiment of the invention, the compositepolypeptide is a single chain polypeptide, meaning that all amino acidsare present in a single, peptide bonded polypeptide chain. Thisembodiment has the advantage that production of a desired single chainpolypeptide product may be achieved very efficiently, since properproduct conformation will depend on establishment of only the necessarysecondary and tertiary polypeptide structures; quaternary polypeptidestructure, in which separate polypeptides exhibiting a certain tertiarystructure associate intermolecularly, need not be considered when theexpressed composite polypeptide is a single chain composite polypeptide.

According to a further embodiment of the invention, the EED may belocated at the C-terminal end or the N-terminal end of the compositepolypeptide. Each location entails the intended advantages describedhereinabove, especially the observed increase in total amount ofexpressed composite polypeptide. A C-terminally located EED will beexpressed last, i.e. after the desired polypeptide, with the effect thatany disulfide bonds necessary within the desired polypeptide will havetime to form as necessary for protein stabilization prior to thetranslation of the EED. This means that when the EED is fullytranslated, the desired disulfide bond between Cys1 and Cys2 within theEED will be the last disulfide bond to form, and that the structure ofthe desired polypeptide will already have been stabilized by anyinternal disulfide bonds. The danger of the formation of undesireddisulfide bonds between Cys1 or Cys2 in the EED portion of the compositepolypeptide and another cysteine residue within the desired polypeptideis therefore minimal.

Positioning the EED at the N-terminal end of the composite polypeptidehas the effect that the nucleic acid encoding the EED will be translatedbefore the nucleic acid encoding the desired polypeptide. This meansthat a disulfide bond between Cys1 and Cys2 within the EED is likely toform before any other cysteine residue within the desired polypeptide istranslated. Again here, the danger of an undesired disulfide bondforming between Cys1 or Cys2 in the EED portion of the compositepolypeptide and a cysteine residue within the desired polypeptide isminimal.

Location of the EED at the N-terminal or C-terminal end of the compositepolypeptide may therefore be determined by considerations of where thederivatization moieties to be bound to the EED would be least likely toperturb the biological activity of the desired polypeptide. In this way,a high degree of experimental flexibility is achieved; the experimenterhas the luxury of choosing the location of the EED which will allow thehighest activity of the desired polypeptide in the final derivatizedcomposite polypeptide, without having to sacrifice the advantagesconferred by the presence of the EED, these advantages having beenexplained above.

According to a preferred embodiment of the invention, the EED of thecomposite polypeptide is of the form: -Cys1-(Xaa)n-Cys2-(Pro)m, whereinn is any integer from 2 to 20; m is 0 (zero) or 1; and wherein Xaa isallowed at each position to be any of the naturally occurring aminoacids, wherein preferably at least 75%, even better at least 80 or 90%of the Xaa residues are selected from Gly, Ala, Val, Leu, Ile, Ser, Thr,Met, Tyr, Asn, and Gln. In a preferred embodiment, all Xaa are Gly, Ser,Ala, or Thr. By allowing the variable n to range from 2, preferably 3,to 20, the linker between Cys1 and Cys2 remains short enough to promoteformation of a disulfide bond between Cys1 and Cys2, yet long enough toallow the linker to double back on itself to do so. Linker lengths of4-5 have been found to promote disulfide linkages between Cys1 and Cys2especially efficiently, with a linker length of 4 amino acids beingespecially preferred for this purpose. Of the amino acids listed abovein this paragraph, Gly and Ser, both alone and in mixtures, have beenfound to be especially amenable to this purpose. Without being bound bytheory, the inventors believe this to be due to the fact that Gly isboth chemically neutral and small, thereby reducing the propensity ofthe linker to participate in undesired chemical reactions whileretaining a maximum degree of unhindered steric flexibility. The aminoacid Ser is believed to confer, through its hydroxyl group, an adequatemeasure of hydrophilicity which may help in preventing a linker which istoo hydrophobic from engaging in undesired hydrophobic interactions withhydrophobic regions of the desired polypeptide. It should be noted thatin the event that the EED is located at the N-terminal end of thecomposite polypeptide, it may be advantageous that m is 0 (zero).

The present embodiment of the invention also allows a Pro residue to bepeptide-bonded to Cys2 at the latter's C-end, although the presence ofPro is not a requirement (i.e. the variable m can also equal zero). Theprovision of Pro has been found to further increase expression yields ofthe composite polypeptide in some circumstances. Without being bound bytheory, the inventors believe that this is due to proline's ability toinhibit proteinase degradation of the composite polypeptide from thelatter's C-terminal end.

In an especially preferred embodiment of the invention, n=4 and (Xaa)4(SEQ ID NO:1) is (Gly)4 (SEQ ID NO:2), (Gly)3Ser (SEQ ID NO:3),(Gly)2SerGly (SEQ ID NO:4), GlySer(Gly)2 (SEQ ID NO:5) or Gly(Ser)3 (SEQID NO:6). In another especially preferred embodiment of the invention,n=5 and (Xaa)5 (SEQ ID NO:7) is (Gly)5 (SEQ ID NO:8), (Gly)4Ser (SEQ IDNO:9), (Gly)3SerGly (SEQ ID NO:10), (Gly)2Ser(Gly)2 (SEQ ID NO:11),GlySer(Gly)3 (SEQ ID NO:12) or Ser(Gly)4 (SEQ ID NO:13). The specialadvantages of using Gly and Ser, both alone and together, in the linkerof the EED have been discussed above. As mentioned above, a linkerlength of 4 amino acids in total was found to lead to the most efficientformation of a disulfide peptide loop between Cys1 and Cys2.

In a further embodiment of the invention, the EED is of the form(His)j-Cys1-(Xaa)n-Cys2-(Pro)m (SEQ ID NO:14) orCys1-(Xaa)n-Cys2-(His)j-(Pro)m (SEQ ID NO:15) wherein j is any integerfrom 2 to 15, and wherein Xaa, n and m are as defined above.Incorporation into the EED of a poly-His sequence (“His-tag”) to theN-side of Cys1 or to the C-side of Cys2 entails several advantages.First, as is known in the art (Porath, J., et al. (1975) Nature 258,598-9; Sulkowski, E. (1985) Trends in Biotech 3, 1-12), a His-tag can bean invaluable tool in the isolation of expressed polypeptide via animmobilized nickel column as well as in the subsequent detection of thepolypeptide. But perhaps more advantageous for the composite polypeptideof the present invention is the special effect that the His-tag has onthe desired formation of the disulfide linkage between Cys1 and Cys2,and thus on the total amount of composite polypeptide obtained inexpression. This effect is especially pronounced when the EED is locatedC-terminal to the desired polypeptide with the His-tag N-terminal ofCys1; or when the EED is located N-terminal to the desired polypeptidewith the His-tag C-terminal of Cys2—in each case the His-tag is locatedat the interface of the EED and the desired polypeptide. Without beingbound by theory, the inventors believe that this special effect may beexplained as follows: Histidine typically bears a positive charge, sothe individual histidine residues in a repeating histidine motif tend tobe electrostatically repelled from one another, leading to an extendedpolypeptide chain in the region of the histidine residues. By placingthis histidine motif within the EED at the interface between the desiredpolypeptide and the EED, these two components of the compositepolypeptide become extended as far away from one another as the lengthof the His-tag allows. This has the effect of reducing the likelihood ofunwanted interactions between the portion of the EED comprising Cys1 andCys2 on the one hand, and the desired polypeptide on the other. At thesame time, by physically separating the EED from the desiredpolypeptide, the likelihood that Cys1 and Cys2 will form a disulfidebond with one another is increased. This is because Cys1 and Cys2 existin this scenario in more or less physical isolation from the rest of thenascent composite polypeptide; in the absence of any other sulfhydrylgroups competing with Cys1 or Cys2 for formation of a disulfide bond, adisulfide bond is more likely to form in the desired fashion between therespective sulfhydryl groups on Cys1 and Cys2.

In an especially preferred embodiment of the invention, j=6, i.e. theEED is of the form (His)6-Cys1-(Xaa)n-Cys2-(Pro)m (SEQ ID NO:16) orCys1-(Xaa)n-Cys2-(His)6-(Pro)m (SEQ ID NO:17), wherein Xaa, n and m areas defined above.

According to a further embodiment of the invention, the derivatized Cys1and/or Cys2 is the reaction product of the Cys1 and/or Cys2 residue/swith a derivatization moiety comprising, e.g., a maleimide group, asulfhydryl group, or a pyridyl disulfide group. All these chemicalgroups react covalently with sulfhydryl. The advantage of thisembodiment of the present invention is that the majority ofderivatization moieties which would be of interest for use inderivatizing the composite polypeptide are available in a formfunctionalized with one of the above groups. As such, the compositepolypeptide of the invention can be derivatized with a wide variety ofvarious reagents for various therapeutic and/or diagnostic purposes. Ofthe groups mentioned above, a maleimide group is especially preferred.The maleimide group reacts nearly completely with sulfhydryl under mildreaction conditions which would not likely damage the desiredpolypeptide in the composite polypeptide, and results in a robustcovalent chemical bond between the sulphur atom of cysteine and one ofthe two unsaturated carbon atoms in the ring of the maleimide group.

In an especially preferred embodiment of the invention, thederivatization moiety comprising a maleimide group is chosen fromPEG-maleimide (“PEG-MAL”), a maleimide-functionalized fluorescencemarker, a maleimide-functionalized assay detection marker, amaleimide-functionalized radioactive tracer, a maleimide-functionalizedprotein crosslinker, a maleimide-functionalized chemotherapeutic agentor a maleimide-functionalized toxin, for example amaleimide-functionalized immunotoxin. Suitable examples of PEG-MAL aremethoxy PEG-MAL 5 kD; methoxy PEG-MAL 20 kD; methoxy (PEG)2-MAL 40 kD;methoxy PEG(MAL)2 5 kD; methoxy PEG(MAL)2 20 kD; methoxy PEG(MAL)2 40kD; or any combination thereof. Any of these reagents may be used asderivatization moieties to confer the known advantages of PEGylation,including increasing the serum half time and reducing theimmunogenicity, of the composite polypeptide of the invention. Suitableexamples of a maleimide-functionalized fluorescence marker arebiotin-maleimide and digoxygenin-maleimide. A suitable example of amaleimide-functionalized radioactive tracer is DTPA-maleimide. Asuitable example of a maleimide-functionalized crosslinker is anN-hydroxysuccinimidyl-maleimide crosslinking species which reactsthrough its N-hydroxysuccinimidyl portion with a free amino group ofanother chemical species to be coupled, and through its maleimideportion with at least one of Cys1 and Cys2 on the composite polypeptideof the invention. The crosslinking species may advantageously be used toeffect, through its N-hydroxysuccinimidyl portion, e.g. a glycosylation,a silylation or a pectinylation of the composite polypeptide of theinvention.

According to a further embodiment of the invention, Cys1 and/or Cys2 isderivatized with a derivatization moiety comprising a sulfhydryl group,in particular wherein said derivatization moiety is Cys2 coupled to Cys1by a disulfide bond. The scenario in which Cys1 forms a disulfide bondwith Cys2 is discussed above. A further example of a derivatization ofCys1 or Cys2 with a derivatization moiety comprising a sulfhydryl groupis when the derivatization moiety is a polypeptide or protein other thanthe composite polypeptide of the invention, and the derivatization isaccomplished by formation of a disulfide bond between, on one side, Cys1and/or Cys2 of the composite polypeptide of the invention and, on theother side, with a Cys residue of the other polypeptide or protein. Afurther possibility of a derivatization moiety comprising a sulfhydrylgroup is a derivatization moiety comprising a 5-thio-2-nitrobenzoic acid(“TNB-thiol”) group.

According to a further embodiment of the invention, both Cys1 and Cys2are derivatized with derivatization moieties. This leads to twoderivatization moieties per inventive composite polypeptide molecule.Such derivatization might be especially advantageous when derivatizing acomposite polypeptide intended for use as an imaging reagent. This isbecause double-derivatization per composite polypeptide would lead to animaging signal twice as intense as would result using a compositepolypeptide derivatized with only one derivatization moiety permolecule. Double-derivatization per composite polypeptide is alsoenvisioned as being advantageous under certain circumstances in whichthe composite polypeptide is intended for use as a therapeutic agent.For instance, if it is desired to PEGylate the composite polypeptideprior to therapeutic administration and a total molecular weight due toPEG of 40 kD is desired, it may prove more advantageous to derivatizethe composite polypeptide at Cys1 and Cys2 with two respective moleculesof 20 kD PEG-MAL than to derivatize only at Cys1 or Cys2 with onemolecule of 40 kD PEG-MAL. Generally, derivatization at each of Cys1 andCys2 can be accomplished by reacting the composite polypeptide of theinvention with a molar excess of derivatization moiety.

According to a further embodiment, either Cys1 or Cys2 is derivatizedwith a first derivatization moiety, while the respective other of Cys2and Cys1, respectively, is derivatized with a second derivatizationmoiety, wherein the second derivatization does not exhibit anyfunctionality other than to block/protect the Cys residue to which it isbound. Conversely to the scenario described above, it may sometimes beadvantageous or necessary to derivatize the composite polypeptide of theinvention only once, for example, when using the composite polypeptideas a diagnostic reagent in a situation where a 1:1 correlation is neededbetween the biological activity of the desired polypeptide within thecomposite polypeptide and the signal measured. Similar scenarios can beenvisioned in which, say, it would be desirable or necessary to PEGylatea composite polypeptide of the invention at only one position. In suchcases, the composite polypeptide can be advantageously incubated undermild reducing conditions sufficient to reduce the disulfide bondexisting between Cys1 and Cys2, but not any other disulfide linkagesexisting throughout the structure of the desired polypeptide tostabilize the latter's structure. Such selective reduction under mildconditions will typically be possible, since disulfide bonds involved instabilization of polypeptide structure will normally be buried withinthis polypeptide structure and therefore poorly accessible by reductionagents in solution, whereas the more exposed, C-terminal EED willgenerally be more accessible. Following opening of the disulfide bondwithin the EED, the composite polypeptide of the invention can then bereacted with the desired first derivatization moiety such that the molaramount of first derivatization moiety is equal to or slightly less thanthe molar amount of inventive composite polypeptide. Precisestoichiometric adjustment of this ratio may be necessary depending onthe first derivatization moiety used, but such adjustment lies wellwithin the ambit of the skilled practitioner's expertise.

Following reaction of either Cys1 or Cys2 with the first derivatizationmoiety, the singly-derivatized composite polypeptide may be isolated bystandard techniques and advantageously subjected to a further reactionwith a second derivatization moiety. The function of the secondderivatization moiety is to deactivate the remaining free sulfhydrylgroup of the underivatized cysteine residue within the EED. To ensurethat the reaction with the second derivatization moiety is efficient,this reaction should advantageously be performed in a molar excess ofsecond derivatization moiety to composite polypeptide. In this sense, asecond derivatization moiety may be any moiety which will reactcovalently with the remaining free cysteine residue within the EED, andmay employ any of the coupling chemistries mentioned above in thecontext of the first derivatization moiety. Since the function of thesecond derivatization moiety is merely to render the remaining cysteineresidue within the EED permanently unreactive, the second derivatizationmoiety should not interfere with the intended activity of the desiredpolypeptide or the first derivatization moiety connected to the othercysteine residue in the EED. For this reason, the second derivatizationmoiety should be chemically and electrostatically inert and as small aspossible. An especially preferred second derivatization moiety isethyl-maleimide. This second derivatization moiety will react with thefree sulfhydryl group of the remaining cysteine residue in the EED toform a covalent C-S bond in the matter already described above.

According to a further embodiment of the invention, the desiredpolypeptide may be any polypeptide for which adequate expression isdesired. This includes all protein and polypeptide molecules of varioussizes (i.e. molecular weights), irrespective of isoelectric points,primary amino acid sequence or desired posttranslational modificationssuch as for example glycosylation or phosphorylation. The desiredpolypeptide may advantageously be a receptor, a ligand or a bindingmolecule. It may be expressed in prokaryotes or in eukaryotes, and mayitself be of natural or recombinant origin.

According to an especially preferred embodiment of the invention, thedesired polypeptide has an even number of cysteine residues required forstabilization of polypeptide structure. This will normally be the case,especially when the desired polypeptide is a single chain polypeptide(i.e. will not interact with any other polypeptide chain followingexpression to form a multichain polypeptide product), since eachdisulfide bond required for stabilizing polypeptide structure requiresthat two cysteine residues be present.

According to an especially preferred embodiment of the invention, thedesired polypeptide is a binding molecule in the form of an antibody.Encompassed within the meaning of “antibody” within this embodiment ofthe invention are single chain mono- and bispecific antibodies, as wellas antibodies. comprising multiple polypeptide chains, such asimmunoglobulin molecules (in which it may be advantageous to expresseach constituent polypeptide chain thereof with an EED of its own) ordiabodies (in which two scFv molecules, each with an EED of its own,associate linearly head-to-tail to form a molecular species capable ofbinding two distinct antigens). Such immunoglobulin molecules may beeither monospecific (i.e. each of the two binding arms of theimmunoglobulin bind to the same antigen) or bispecific (i.e. each of thetwo binding arms of the immunoglobulin bind to different antigens), forexample such bispecific immunoglobulins as would be obtained from ahybrid-hybridoma.

In an especially preferred embodiment of the invention, the desiredpolypeptide is a monospecific single chain antibody. Within the meaningof the present invention, the term “monospecific single chain antibody”may be understood as a single polypeptide chain comprising at least oneantibody variable region. This at least one antibody variable region maybe present in nature, for example in an antibody library of naturalorigin, or may be synthetic in that it comprises elements found in orderived from nature, but these elements are present in combinations notpresent as such in nature. Alternatively, a monospecific single chainantibody may comprise both natural and synthetic elements. Specificallyfalling within the meaning of the term “monospecific single chainantibody” are single domain antibodies, scFv molecules, as well ashumanized and/or deimmunized variants thereof.

According to a further especially preferred embodiment of the invention,the desired polypeptide may be a bispecific single chain antibody.Within the meaning of the present invention, the term “bispecific singlechain antibody” may be understood as two monospecific single chainantibodies as described above existing on a single polypeptide chain,and preferably separated from one another by a suitable polypeptidespacer sequence. Examples of such spacers may be found e.g. in EP 623679B1 and U.S. Pat. No. 5,258,498. As such, the composite polypeptide mayadvantageously represent a derivatized bispecific antibody.

According to a further embodiment of the invention, the bispecificsingle chain antibody comprises a first monospecific single chainantibody (first binding portion) specifically binding to an effectorantigen and a second monospecific single chain antibody (second bindingportion) specifically binding to a target antigen. This generalconstruction has the advantage that the desired polypeptide canspecifically bind with its first binding portion to an effector antigensuch that the effector antigen bound for example becomes activated. Thebiological activity triggered by this effector antigen may then bedirected to, for example, a cell bearing the target antigen, to whichthe second portion of the bispecific single chain antibody specificallybinds. Here, it is to be understood that the terms “first” and “second”imply no restriction with respect to the location of the antibodyportions relative to the N-terminus or C-terminus of the polypeptide. Itis therefore within the ambit of this embodiment of the invention thatthe composite polypeptide comprises a desired polypeptide in which thefirst binding portion specifically binding to the effector antigen maybe located towards the desired polypeptide's N-terminal end orC-terminal end.

In an especially preferred embodiment of the invention, the effectorantigen is chosen from the CD3 antigen, the CD64 antigen, the CD89antigen and the NKG2D antigen. In another preferred embodiment of theinvention, the target antigen is chosen from EpCAM, CCR5, CD19, HER-2neu, HER-3, HER-4, EGFR, PSMA, CEA, MUC-1 (mucin), MUC2, MUC3, MUC4,MUC5AC,MUC5B, MUC7, hCG, Lewis-Y, CD20, CD33, CD30, ganglioside GD3,9-O-Acetyl-GD3, GM2, Globo H, fucosyl GM1, Poly SA, GD2, CarboanhydraseIX (MN/CA IX), CD44v6, Sonic Hedgehog (Shh), Wue-1, Plasma Cell Antigen,(membrane-bound) IgE, Melanoma Chondroitin Sulfate Proteoglycan (MCSP),CCR8, TNF-alpha precursor, STEAP, mesothelin, A33 Antigen, Prostate StemCell Antigen (PSCA), Ly-6; desmoglein 4, E-cadherin neoepitope, FetalAcetylcholine Receptor, CD25, CA19-9 marker, CA-125 marker andMuellerian Inhibitory Substance (MIS) Receptor type II, sTn (sialylatedTn antigen; TAG-72), FAP (fibroblast activation antigen), endosialin,EGFRvIII, LG, SAS and CD63. Here, all the above antigens (both effectorand target antigens) may be human antigens.

In a very preferred embodiment of the invention, the target antigen isthe human CD19 antigen, while the effector antigen is the human CD3antigen. As such, this embodiment provides for a derivatized compositepolypeptide capable of directing the cytotoxic potential of cytotoxic Tcells against B lymphocytes bearing the CD19 antigen. Such a medicationhas great potential as a therapeutic agent in the treatment of B cellmalignancies. As a result, it is of great interest to derivatize such acomposite polypeptide in its EED with one or more PEG molecules in orderto increase the serum half time while simultaneously reducing theimmunogenicity of the composite polypeptide.

In another very preferred embodiment of the invention, the targetantigen is the human EpCAM antigen, while the effector antigen is thehuman CD3 antigen. As such, this embodiment provides for a derivatizedcomposite polypeptide capable of directing the cytotoxic potential ofcytotoxic T cells against cells bearing the EpCAM antigen. The EpCAMantigen is expressed in many human malignant cells; such a derivatizedcomposite polypeptide therefore has great potential in the treatment ofa wide spectrum of human cancers. As with the anti-CD3xanti-CD19composite polypeptide described above, it is also of great interest toderivatize such an anti-CD3xanti-EpCAM composite polypeptide in its EEDwith one or more PEG molecules.

A further aspect of the invention relates to a composition comprisingany of the composite polypeptides described above and a pharmaceuticallyacceptable carrier.

In a further aspect, the invention provides a method of producing acomposite polypeptide, wherein the composite polypeptide comprises adesired polypeptide and is expressed in higher yield than the desiredpolypeptide, said method comprising

-   -   a) providing a nucleotide sequence encoding the desired        polypeptide;    -   b) incorporating on either end of the nucleotide sequence        encoding the desired polypeptide a nucleotide sequence encoding        an expression enhancing domain (“EED”) encoding, said nucleotide        sequence encoding the EED comprising codons for first and second        cysteine amino acid residues Cys1 and Cys2, respectively, the        codon for Cys1 being located closer to the 5′-end of the        nucleotide sequence than the codon for Cys2, wherein the codons        for Cys1and Cys2 are separated by a nucleotide sequence encoding        a polypeptide linker, said linker being cysteine-free; and        defining a length sufficient to allow Cys1 and Cys2 to engage in        an intramolecular disulfide bond with one another;    -   c) transfecting the nucleotide sequence from step (b) into a        host expression system in a suitable vector;    -   d) incubating the host expression system under conditions        suitable to result in expression of the nucleotide sequence from        step (b);    -   e) isolating the polypeptide expressed in step (d) to obtain the        composite polypeptide.

A preferred embodiment of this aspect of the invention comprises thefurther step of derivatizing the composite polypeptide obtained in step(e) at Cys1 and/or Cys2. Such derivatization may be performed asdescribed above, namely by reducing the intramolecular disulfide bondbetween Cys1 and Cys2 (this intramolecular disulfide bond itself beingseen as a derivatization) under reducing conditions (for example usingdithiothreitol, or DTT), followed by reaction of the reduced productwith another derivatization moiety bearing a chemical group which reactswith at least one of the free thiol groups of Cys1 and Cys2.

The invention will now be described in more detail by way of thefollowing nonlimiting figures and examples.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1 Antigen-specific ELISA as dependent on linker length between Cys1and Cys2

FIG. 2 Antigen-specific ELISA results as a measure of expression yieldswith one C-terminal cysteine residue, and with two C-terminal cysteineresidues separated by a 4-glycine linker

FIG. 3 Western blot results as a measure of expression yields with oneC-terminal cysteine residue, and with two C-terminal cysteine residuesseparated by a 4-glycine linker (1. and 2. (SEQ ID NO:18); 3. and 4.(SEQ ID NO:19); 5. and 6. (SEQ ID NO:20)

FIG. 4 Gel-filtration chromatography results showing an elution profilefrom a composite polypeptide according to the invention and an elutionprofile from a polypeptide with only one C-terminal cysteine residue

FIG. 5 SDS-PAGE of the monomer and dimer scFv species obtained bygel-filtration chromatography; Gel results under non-reducing (left) andreducing (right) conditions are shown (1. and 2. (SEQ ID NO:19); 3. and4. (SEQ ID NO:20)

FIG. 6 SDS-PAGE of scFv with one and two C-terminal cysteine residues,before and after reaction with 20 kD PEG-maleimide (1. and 3. (SEQ IDNO:19); 2. and 4. (SEQ ID NO:20).

The invention will now be described in further detail by way of thefollowing, non-limiting examples.

EXAMPLES Example 1 Cloning and Expression of scFv with a C-terminal(His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) tag (i.e. scFv with an EED asdefined above)

An scFv molecule, i.e. a polypeptide unifying heavy and light chainantibody variable regions and a (Gly4Ser)3 (SEQ ID NO:30) polypeptidelinker disposed therebetween, was used as a model molecule fordemonstrating the concept of the invention. This scFv specifically bindsto a predetermined antigen, subsequently referred to as “Antigen”. AnscFv with a C-terminal (His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) tag wasconstructed by a PCR-reaction with VL-specific primers, whereas the scFvnucleotide sequence was independently extended with each of therespective nucleotide sequences of the (His)6-Cys-(Gly)x-Cys-Pro motif(A: TGCGGTGGCTGCCCGTAA (SEQ ID NO:21), B: GCGGTGGCGGTTGCCCGTAA (SEQ IDNO:22), C: TGCGGTGGCGGTGGCTGCCCGTAA (SEQ ID NO:23), D:TGCGGTGGCGGTGGCTGCCCGTAA) (SEQ ID NO:24). This yielded four nucleotidesequences encoding 4 separate scFvs, each with a C-terminal tag havingtwo cysteine residues separated by glycine linkers of varying length.The His-Tag was employed in later detection and purification steps. Theresulting VL fragments were subcloned via the restriction enzymerecognition sites SalI and NotI (introduced by PCR) into pBADpelB(derived from the vector pBADMycA-His from Invitrogen) containing thecorresponding VH behind a pelB leader sequence for periplasmicexpression. After transformation into heat shock competent E.coliXL1Blue, a single clone was cultivated in selective media (LB 50μg/mlCarbenicillin) and the plasmid was prepared according to standardprotocols. Successful cloning was confirmed by sequencing the insert(Sequiserve, Munich).

E.coli BL21DE3 were transformed with the expression plasmid coding forthe respective scFv with one or two C-terminal Cys residues and grown onselective agar. One colony was used to inoculate 5 ml LB 50 g/mlCarbenicillin over night at 37° C. For the production culture, 500 ml SBgrowth medium containing 20 mM MgC12 and 50 μg/ml Carbenicillin in 2 1shaker flasks were inoculated with the bacterial suspension of theovernight culture and further incubated at 37° C. to an optical densityat OD600 of 0.6-0.8. Protein production was induced by addingL-arabinose to a final concentration of 0.2% and reduction of thetemperature to 30° C. After a four hour production phase at 30° C. thebacteria were harvested and resuspended in 40 ml PBS. Through fourrounds of freezing at −70° C. and thawing at 37° C. the outer membranewas destroyed by temperature shock and the soluble periplasmic proteinsincluding the scFv-fragments were released into the liquid. Afterelimination of intact cells and cell debris by centrifugation, thesupernatant was used for ELISA analysis.

ELISA analysis of the periplasmic preparation was carried out using anELISA-plate (Nunc MaxiSorp) coated with ProteinL (2μg/ml in PBS).Coating was performed overnight at 4° C. After washing with PBS 0.05%Tween, the plate was blocked with 100 μl PBS containing 3% BSA for 1h atroom temperature. After washing, 50 μl periplasm were added, dilutedserially 1:3 and incubated for 1 h at room temperature. After anadditional washing step, detection of scFv bound to ProteinL was carriedout specifically using 50 μl Antigen-Biotin (1.5 μg/ml containing PBS 1%BSA) detected by streptavidin-HRP (Dako, 1 μg/ml in PBS containing 1%BSA). The signal was detected by adding 100 μl ABTS(2,2′-Azino-di[3-ethylbenzthiazoline sulfonate (6)] diammoniumsalt)-substrate solution for 15-30 min. The OD-values were measured onan ELISA reader at a wavelength of 405 nm. The results are shown in FIG.1, in which “HCP”, CH2GlyCP″, “HC3GlyCP”, “HC4GlyCP” and “HC5GlyCP”respectively refer to scFv molecules with C-terminal tags containing(His)6-Cys-Pro (SEQ ID NO:19), (His)6-Cys-(Gly)2-Cys-Pro (SEQ ID NO:25),(His)6-Cys-(Gly)3-Cys-Pro (SEQ ID NO:26), (His)6-Cys-(Gly)4-Cys-Pro (SEQID NO:20) and (His)6-Cys-(Gly)5-Cys-Pro (SEQ ID NO:27). As can be seenin FIG. 1, the highest yield of scFv binding to Antigen was observed forthe construct with four glycines used as linker between the twoC-terminal cysteines.

Example 2 Confirmation of the Higher Protein Yield of scFv with(His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) tag (i.e. an EED, as definedabove) as compared to the scFv with (His)6-Cys-Pro (SEQ ID NO:19) tag(i.e. without an EED as defined above).

Protein expression levels of the scFv extended with(His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) tag (i.e. seFv with the EED asdefined above, referred to as “4Gly” in FIG.2) and the scFv extendedwith the (His)6-Cys-Pro (SEQ ID NO:19) C-terminal tag (i.e. scFv withoutthe EED as defined above, referred to as “HCP” in FIG.2) were compared.Both constructs were analyzed on small scale using the E.coli strainBL21DE3. In each case 10 different colonies were inoculated in 5 mlSB/20mM MgCl2/50 μg/ml Carbenicillin for four hours at 37° C. in ashaking incubator. Again protein production was initiated by theaddition of 0.2% L-arabinose to the cell cultures and a temperaturedecrease to 30° C. After an overnight induction period cells wereharvested, resuspended in 1 ml PBS and the periplasmic fraction wasisolated by the freeze/thaw method and analyzed in an Antigen-specificELISA as described in Example 1. The results of this analysis are shownin FIG. 2. The ELISA results show clearly the significantly increasedyield of scFv with the (His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) tag(“4Gly”) in the crude periplasm as compared to the scFv speciescontaining a C-terminal (His)6-Cys-Pro (SEQ ID NO:19) motif, but lackinga second cysteine residue. Clearly, then, the ability to form acontrolled, intramolecular disulfide bond between the two cysteineresidues in the C-terminal tag (i.e. the EED as defined above) is ofcrucial importance for achieving the enhanced production yieldsobserved.

The periplasmic fractions were further analyzed by non-reducing SDS-PAGEfollowed by Western blot techniques according to standard protocols. Thedetection of the His-tagged scFv was accomplished using an anti-pentaHis antibody, Qiagen (1 μg/ml in PBS containing 0.1% BSA) detected withan alkaline phosphatase-conjugated goat anti-mouse antibody, Sigma (1μg/ml in PBS containing 0.1% BSA). The protein blot was developed byadding BCIP/NBT substrate solution (Sigma, B-1911). The results areshown in FIG. 3.

Lanes 1 and 2 of the Western blot shown in FIG. 3 show bands of scFvwith (His)6-Pro (SEQ ID NO:28) at the C-terminal end. The intensity ofscFv bands in the Western blot—and therefore the amount of totalpolypeptide expressed—is seen to decrease drastically in lanes 3 and 4,corresponding to scFv with (His)6-Cys-Pro (SEQ ID NO:19) at theC-terminal end of the polypeptide. Lanes 5 and 6, corresponding to scFvwith (His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) at the C-terminal end ofthe polypeptide, show a band intensity which is once again comparable tothe intensity seen in lanes 1 and 2. This clearly demonstrates that theloss in protein expression suffered in adding a single cysteine residueto the C-terminal end of the scFv polypeptide (lanes 3 and 4) wasregained by adding a second cysteine residue, separated from the firstcysteine residue by a polypeptide linker allowing disulfide bondformation between the two cysteine residues (lanes 5 and 6).

Taken together, the results of the ELISA (FIG. 2) and the Western blot(FIG. 3) analyses show clearly the higher protein/scFv yield of the scFvconstruct with two C-terminal cysteine residues as compared to the scFvconstruct with only one C-terminal cysteine.

Example 3 Purification of scFv with the (His)6-Cys-(Gly)4-Cys-Pro (SEQID NO:20) tag (i.e. the EED, as defined above)

E. coli BL21DE3 were transformed with the expression plasmid and grownon selective agar. A single colony was used to inoculate 5 ml LB 50μg/ml Carbenicillin overnight at 37° C. For the production culture, 500ml SB/20 mM MgC12/50 μg/ml Carbenicillin in 2 1 shaker flasks wereinoculated with the bacterial suspension of the overnight culture andgrown at 37° C. to an optical density at OD600 of 0.6-0.8. Proteinproduction was induced by adding L-arabinose to a final concentration of0.2% and reduction of the temperature to 30° C. After an overnightproduction phase at 30° C. the bacteria were harvested and resuspendedin 40 ml PBS. The outer membrane was destroyed by temperature shock andthe soluble periplasmic proteins including the scFv-fragment werereleased into the liquid. After elimination of intact cells and celldebris by centrifugation, the supernatant was further purified.

SCA molecules were initially purified by an IMAC affinity columninteracting with the C-terminal His-Tag. This was performed using aQiagen Ni-NTA superflow column according to the protocol provided by themanufacturer. The column was equilibrated with 20 mM sodium phosphate0.4 M NaCl, pH 7.2 and the periplasmic preparation (40 ml) was appliedto the column at a flow rate of 2 ml/min. Afterwards the column waswashed with 5 column volumes of equilibration buffer containing 0.025 Mimidazole to remove unbound sample. Elution was carried out usingequilibration buffer containing 0.5 M imidazole in 5 column volumes.Eluted protein fractions were pooled for further purification steps.

To achieve a separation of the molecular weight, i.e. separation intomultimeric, dimeric and monomeric fractions, gel filtrationchromatography was performed on a superdex S75 prep grade columnequilibrated with PBS (Gibco). Eluted protein monitored by continuousmeasurement of 280 nm light absorption (flow rate 1 ml/min) weresubjected to standard SDS-PAGE. The results are shown in FIG. 4. FIG. 4shows two elution profiles A and B, the lower one (profile A) being theelution profile of scFv with a C-terminal (His)6-Cys-Pro (SEQ ID NO:19)motif, the higher one (profile B) being the elution profile of scFv witha C-terminal (His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) motif (i.e. scFvwith the EED as defined above). As can clearly be seen, incorporation ofa second cysteine residue in the C-terminal portion of the scFv inadequate separation from the first cysteine residue for the formation ofa disulfide loop leads not only to a higher monomer:dimer product ratio,but also to a higher overall protein yield irrespective of monomer ordimer isoform.

This obvious optical analysis is corroborated by calculation of theisoform concentrations. The protein concentrations were calculated usingthe AUC value (determined by the UNICORN software) and thesequence-specific extinction coefficient. The concentration valuesobtained are summarized below in Table 1:

TABLE 1 Calculated Sample Total protein Total Amino Acid SequencePolypeptide concentration volume per isoform protein of C-terminalportion isoform (μg/ml) (ml) (μg) (μg) HHHHHHCP Monomer 14.2 6 85.2276.6 (SEQ ID NO: 19) HHHHHHGP Dimer 31.9 6 191.4 (SEQ ID NO: 19)HHHHHHCGGGGCP Monomer 69 8 552 775.2 (SEQ ID NO: 20) HHHHHHCGGGGCP Dimer27.9 8 223.2 (SEQ ID NO: 20)

From the above table, the following statements can be made. First, themonomer:dimer ratio for scFv with a single cysteine residue in itsC-terminal portion is about 1:2.25. By addition of a second cysteine tothe C-terminal portion of the scFv and by disposing a suitable linkerbetween the first and second cysteine residues, an intramoleculardisulfide bond is promoted, and the monomer:dimer ratio of obtained scFvis increased about 5.5 fold, to 1:0.4. Viewed from the standpoint ofoverall polypeptide yield irrespective of polypeptide isoform, theincrease in yield from 276.6 μg for the scFv with a single cysteineresidue to 775.2 μg for the scFv with two cysteine residues representsan increase in overall protein expression of about 280%, or almost3-fold.

Analysis of the gel-filtered monomeric and dimeric fraction by SDS-PAGEunder non-reducing and reducing conditions (FIG. 5) showed clearly thatapproximately 80% of the dimeric fraction of the scFv with a singlecysteine in its C-terminal portion are dimeric, crosslinked by disulfidelinkage (FIG. 5, non-reducing gel, lane 2), whereas the dimeric fractionof the scFv with two cysteine residues in its C-terminal portion existsmainly as a monomer, due to the formation of a disulfide loop betweenthe two C-terminal cysteine residues (FIG. 5, non-reducing gel, lane 4).The dimer disaggregation into monomer (FIG. 5, non-reducing gel, lane 4)is an indication that aggregation had occurred only by protein-proteininteraction and was not due to disulfide crosslinking. Lanes 1 and 3 ofFIG. 5 (reducing and non-reducing conditions) show the correspondingmonomer fractions.

The same samples were also run on a reducing gel (FIG. 5). Regarding thescFv without the (His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) tag, lanes 2shows that any dimer present in the non-reducing gel was in fact due tothe formation of unwanted disulfide linkages between cysteine residuesin two respective polypeptide molecules. The same holds true for theresidual minimal amount of dimer of the scFv with the(His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) tag (lane 4). The reducingconditions within the reducing gel suffice to open these disulfidelinkages so that the only bands observed are the monomeric species ofthe scFv polypeptide in which no cysteine residue within the scFvs areable to form disulfide linkages with any other cysteine residue.

Example 4 Side directed PEGylation of scFvs with and without theC-Terminal (His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) tag

PEGylation at free cysteines should result in a stable homogeneousscFv-PEG conjugate. Two protein solutions containing, respectively,purified scFv with the C-terminal (His)6-Cys-(Gly)4-Cys-Pro tag and scFvwith the C-terminal (His)6-Cys-Pro (SEQ ID NO:19) tag were incubatedwith DTT at a final concentration of 2 mM for one hour at roomtemperature to reduce the terminal disulfide bridge, resulting in twofree sulfhydryl groups.

Gel filtration (Sephadex G25 M, Amersham) of each polypeptide separatelyto remove residual DTT was then performed using PBS as a running buffer.mPEG-Maleimide MW 20 kD (Shearwater, 2D2M0P01) was added to the firsthalf of each polypeptide sample in a 10-fold molar excess of PEGmolecules. The term “mPEG” here carries the known meaning, namely“methoxy polyethylene glycol”. The other half of each polypeptide samplewas incubated with a 10-fold molar excess of ethylmaleimide (Sigma,E-1271) as a control.

Each reaction was allowed to occur for 2 hours with agitation at roomtemperature. All samples were analyzed by SDS-PAGE under non-reducingconditions and stained with silver according to standard protocols(Invitrogen, Cat. No. LC6100). The results are shown in FIG. 6.

Lane 1 of FIG. 6 depicts scFv with (His)6-Cys-Pro (SEQ ID NO:19) at itsC-terminal end. The cysteine residue has been blocked by reaction withethylmaleimide. Lane 2 of FIG. 6 shows an scFv with(His)6-Cys-(Gly)4-Cys-Pro (SEQ ID NO:20) at its C-terminal end, in whichboth cysteine residues have been blocked by reaction withethylmaleimide. The relative intensities of the bands in lanes 1 and 2(i.e. the band in lane 2 is much more intense than the band at the sameposition in lane 1) is a measure of the enhanced expression efficiencyachieved when expressing the scFv with two C-terminal cysteine residuesas compared to that achieved when expressing the scFv with a singleC-terminal cysteine residue.

Lane 3 of FIG. 6 shows the result of coupling an scFv with a singleC-terminal cysteine residue with PEG-maleimide of 20 kD molecularweight. As can be seen in the upper portion of lane 3, only a very faintband of PEG-coupled scFv was obtained, the faintness of which is likelyan indication of the poor expression yields, and therefore less absoluteamounts of scFv obtained using scFv with only a single C-terminalcysteine residue. In sharp contrast, lane 4 of FIG. 6, in which the scFvwith two C-terminal cysteine residues separated from one another by a4-glycine linker has been reacted with 20 kD PEG, shows two distinctbands. One band is at the same molecular weight as the correspondingunreacted species, indicating that the reaction with 20 kD PEG did notproceed to completion. The other, higher band at heavier molecularweight is an indication that 20 kD PEG has reacted with both of the twocysteine residues in the C-terminal portion of the scFv, as it is ofhigher molecular weight than the PEGylation product of the scFv withonly a single C-terminal cysteine residue. It should be emphasized thatit was only possible to obtain sufficient cysteine-containing startingmaterial for the subsequent PEGylation reaction by incorporating notone, but two cysteine residues in the C-terminal portion of the scFv,separated from one another by a 4-glycine linker.

The invention claimed is:
 1. A composite polypeptide, said compositepolypeptide comprising a desired polypeptide and an expression enhancingdomain (“EED”), located at the C- or N-terminal end of the compositepolypeptide, said EED comprising first and second cysteine amino acidresidues Cys1 and Cys2, respectively, Cys1 being located closer to theN-terminus of the composite polypeptide molecule than Cys2, wherein Cys1and Cys2 are separated by a polypeptide linker, said linker: being freeof cysteine and proline; defining a length sufficient to allow Cys1 andCys2 to engage in an intramolecular disulfide bond with one another; andhaving a flexible polypeptide conformation essentially free of secondarypolypeptide structure in aqueous solution, wherein at least one of Cys1and Cys2 is derivatized with a derivatization moiety, and wherein thedesired polypeptide is an antibody.
 2. The composite polypeptide ofclaim 1, wherein at least 75% of the amino acid residues in the linkerare selected from Gly, Ala, Val, Leu, Ile, Ser, Thr, Met, Tyr, Asn, andGln.
 3. The composite polypeptide of claim 1 or 2, wherein the compositepolypeptide is a single chain polypeptide.
 4. The composite polypeptideof claim 1, wherein the EED is of the form: -Cys1-(Xaa)n-Cys2-(Pro)m(SEQ ID NO:29), wherein n is any integer from 2 to 20; m is 0 (Zero) or1; and Xaa is allowed at each position to be Gly, Ala, Thr, or Ser. 5.The composite polypeptide of claim 4, wherein n=4 and (Xaa)4 (SEQ IDNO:1) is (Gly)4 (SEQ ID NO:2), (Gly)3Ser (SEQ ID NO:3), (Gly)2SerGly(SEQ ID NO:4), GlySer(Gly)2 (SEQ ID NO:5) or Gly(Ser)3 (SEQ ID NO:6). 6.The composite polypeptide of claim 4, wherein n=5 and (Xaa)5 (SEQ IDNO:7) is (Gly)5 (SEQ ID NO:8), (Gly)4Ser (SEQ ID NO:9), (Gly)3SerGly(SEQ ID NO:10), (Gly)2Ser(Gly)2 (SEQ ID NO:11), GlySer(Gly)3 (SEQ IDNO:12) or Ser(Gly)4_(SEQ ID NO: 13).
 7. The composite polypeptide ofclaim 4, wherein the EED is of the form:-His-His-His-His-His-His-Cys1-(Xaa)n-Cys2-(Pro)m (SEQ ID NO: 16); or-Cysl -(Xaa)n-Cys2-His-His-His-His-His-His-(Pro)m (SEQ ID NO: 17). 8.The composite polypeptide of claim 1, wherein the derivatized Cys1and/or Cys2 is the reaction product of the Cys1 and/or Cys2 residue/swith a derivatization moiety comprising a maleimide group, a sulfhydrylgroup or a pyridyl disulfide group.
 9. The composite polypeptide ofclaim 8, wherein the derivatization moiety comprising a maleimide groupis selected from PEG-maleimide (“PEG-MAL”), a maleimide-functionalizedfluorescence marker, a maleimide-functionalized assay detection marker,a maleimide-functionalized radioactive tracer or amaleimide-functionalized protein crosslinker.
 10. The compositepolypeptide of claim 9, wherein the PEG-MAL is chosen from: methoxyPEG-MAL 5 kD; methoxy PEG-MAL 20 kD methoxy (PEG)2-MAL 40 kD; methoxyPEG(MAL)2 5 kD; methoxy PEG(MAL)2 20 kD; methoxy PEG(MAL)2 40 kD; or anycombination thereof.
 11. The composite polypeptide of claim 8, whereinCys1 or Cys2 is derivatized with a derivatization moiety comprising a5-thio-2-nitrobenzoic acid (“TNB-thiol”) group or a sulfhydryl group.12. The composite polypeptide of claim 1, wherein both Cys1 and Cys2 arederivatized with derivatization moieties.
 13. The composite polypeptideof claim 1, wherein either Cys1 or Cys2 is derivatized with a firstderivatization moiety, while the respective other of Cys2 or Cys1,respectively, is derivatized with a second derivatization moiety. 14.The composite polypeptide of claim 13, wherein the second derivatizationmoiety is ethyl maleimide.
 15. The composite polypeptide of claim 1,wherein the antibody is selected from a monospecific single chainantibody or a bispecific single chain antibody.
 16. The compositepolypeptide of claim 15, wherein the bispecific single chain antibodycomprises a first portion specifically binding to an effector antigenand a second portion specifically binding to a target antigen.
 17. Thecomposite polypeptide of claim 16, wherein the effector antigen isselected from the human CD3 antigen, the human CD64 antigen, the humanCD89 antigen and the human NKGZD antigen.
 18. The composite polypeptideof claim 16 or 17, wherein the target antigen is selected from the groupconsisting of EpCAM, CCR5, CD19, HER-2 neu, HER-3, HER-4, EGFR, PSMA,CEA, MUC-1 (mucin), MUC2, MUC3, MUC4, MUC5AC, MUC5B, MUC7, hCG, Lewis-Y,CD20, CD33, CD30, ganglioside GD3, 9—O—Acetyl-GD3, GM2, Globo H, fucosylGM1, Poly SA, GD2, Carboanhydrase IX (MN/CA IX), CD44v6, Sonic Hedgehog(Shh), Wue-1, Plasma Cell Antigen, (membrane-bound) IgE, MelanomaChondroitin Sulfate Proteoglycan (MCSP), CCR8, TNE-alpha precursor,STEAP, mesothelin, A33 Antigen, Prostate Stem Cell Antigen (PSCA), Ly-6;desmoglein 4, E- cadherin neoepitope, Fetal Acetylcholine Receptor,CD25, CA19-9 marker, CA-125 marker and Muellerian Inhibitory Substance(MIS) Receptor type II, sTn (sialylated Tn antigen; TAG-72), FAP(fibroblast activation antigen), endosialin, EGERvIII, LG, SAS and CD63,and wherein all said antigens are human antigens.
 19. A compositioncomprising the composite polypeptide of claim 1 and a pharmaceuticallyacceptable carrier.
 20. The composite polypeptide of claim 1 whereinsaid derivization moiety is Cys2 coupled to Cys1 by a disulfide bond.21. A method of producing a composite polypeptide wherein the compositepolypeptide comprises a desired polypeptide and an expression enhancingdomain (“EED”) located at the C- or N-terminus of the compositepolypeptide. said EED comprising first and second cysteine amino acidresidues Cys1 and Cys2, respectively, Cys1 being located closer to theN-terminus of the composite polypeptide molecule than Cys2, wherein Cys1and Cys2 are separated by a polypeptide linker, said linker: being freeof cysteine and proline; defining a length sufficient to allow Cys1 andCys2 to engage in an intramolecular disulfide bond with one another; andhaving a flexible polypeptide conformation essentially free of secondarypolypeptide structure in aqueous solution, wherein at least one of Cys1and Cys2 is derivatized with a derivatization moiety, and wherein thedesired polypeptide is an antibody, and wherein the compositepolypeptide comprising the desired polypeptide is expressed in higheryield than the desired polypeptide, said method comprising; a) providinga nucleotide sequence encoding the desired polypeptide; b) incorporatingon either end of the nucleotide sequence encoding the desiredpolypeptide a nucleotide sequence encoding an expression enhancingdomain (“EED”) encoding, said nucleotide sequence encoding the EEDcomprising codons for first and second cysteine amino acid residues Cys1and Cys2, respectively, the codon for Cys1 being located closer to the5′-end of the nucleotide sequence than the codon for Cys2, wherein thecodons for Cys1 and Cys2 are separated by a nucleotide sequence encodinga polypeptide linker, said linker being cysteine-free; and defining alength sufficient to allow Cys1 and Cys2 to engage in an intramoleculardisulfide bond with one another; c) transfecting the nucleotide sequencefrom step (b) into a host expression system in a suitable vector; d)incubating the host expression system under conditions suitable toresult in expression of the nucleotide sequence from step (b); and e)isolating the polypeptide expressed in step (d) to obtain the compositepolypeptide.
 22. The method of claim 21, further comprising the step ofderivatizing the composite polypeptide obtained in step (e) at Cys1and/or Cys2.